ANATOMICAL CHANGES IN PLUM FRUIT PRUNUS DOMESTICA L. AFTER FRUIT THINNING USING POTASSIUM SALT OF ALPHA-NAPHTALENEACETIC ACID

Aleksander Gonkiewicz, Kazimierz Nosal
Department of Pomology, University of Agriculture in Cracow, Poland

ABSTRACT

The aim of auxin application in thinning of flowers and young fruits is to reduce the number of set fruits and to obtain a high quality fruit yield. Six-year old plum trees ‘Stanley’ and ‘Iroquois’ cultivars treated with a solution of potassium salt of alpha-naphtaleneacetic acid (NAA) after blooming were used in the experiment. The applied concentrations varied from 30 to 160 mg NAA l^-1. Microscopic preparations of flowers and young fruits were made using fluorescence and paraffin methods. There was noted a slight stimulating effect of NAA treatment on the fertilization process. It was also found that the applied auxin induced callose saturation and clogging up of conductive bundles and the chalazal part of the ovule. The inhibition of assimilate translocation to the ovule due to the clogging up of the chalazal part brought about an inhibition in endosperm development and resulted in embryo degeneration.

Key words: callose, NAA, plum, thinning.

Abbreviations: NAA – potassium salt of alpha-naphtaleneacetic acid

INTRODUCTION

Many plum cultivars show a tendency towards excessive fruit setting, which resulting in high yields of very small fruit of low commercial value. The excessive fruit setting also inhibits the formation of flower primordia for the next year bringing about the phenomenon of biennial bearing. It can be prevented by the thinning of flowers and young fruit conducted in the years of intensive fruit setting. For this aim trees are sprayed with various preparations. Almost all available plant hormons and syntetic growth regulations were tested, but NAA has been used the most often during 40 last year [19]. Auxin NAA is especially useful to plum thinning [8], but the role of NAA in mechanism of fruit dropping is not quite clear yet [21]. Several theories concerning the mechanism of NAA effect have been described in the literature. One of them is based on the action of ethylene whose synthesis is stimulated by exogenous auxin [4]. The influence of NAA on intensity of fruit drop mechanism is very changeable and really difficult to predict [19]. The aim of this research was to specify the mechanism of fruit dropping after thinning using potassium salt of alpha-naphtaleneacetic acid.

MATERIAL AND METHODS

Research was carried out in 1999-2001, on six-year old ‘Stanley’ and ‘Iroquois’ plum trees (Prunus domestica L.). The spraying with a solution of potassium salt of alpha-naphthaleneacetic acid (NAA, C₁₂H₉O₂K, Sigma) was conducted by the end of the blooming period, however, the main indication of the time of treatment was a distinctly visible abscission layer between the perianth and the pedicle (phot. 1), signaling the fertilization [11]. In the first year of the experiment with the cultivar ‘Stanley’ concentrations of NAA were 30 and 40 mg l^-1 and in the successive years 30, 40 and 50 mg l^-1. In the second year the cultivar ‘Iroquois’ treated with high concentrations of 40, 80 and 160 mg NAA l^-1 was included in the experiment. Control trees were not treated.

In the investigation of the effect of NAA treatments on the anatomical development of young fruits, microscopic observations of samples prepared using fluorescence microscopy and paraffin methods were carried out. The examined flowers and young fruits of the cultivars ‘Stanley’ and ‘Iroquois’ were sampled from a control plot (untreated) and from plots with trees treated with NAA. The observations were conducted during about two weeks from the time of full blooming, however, the presented results concern four days, beginning from the second day after the auxin treatment.

Fluorescence method. The aim of microscopic examination using the fluorescence method was to determine the consequential effect of NAA on fertilization percentages and the accumulation of callose in the vascular tissue of young fruits. Specimens were fixed with FAA [20] for 24 hours, then were soaked in NaOH 30% for 24 hours and next (without a rinse) in H₂O₂ 6% at 36°C for 1 hour (to bleach) and staining in aniline blue (100 ml distillated water, 2.6 g potassium phosphate tribasic pure, 100 mg aniline blue; pH = 12.3; time of staining about 20 min. in underpressure pump). Smear specimens were examined at fluorescence microscope (Russian microscope МЛ-2, filter уфс 6-5, 310-380 nm) with UV rays according to Martin [12] in modification Lech [10]. Callosa is bright yellow if observed using this method. In one replication were young fruits tested from one day. The statistic calculation consisted results from four days, from second to sixth day after treatment. The growing pollen tubes into micropyle signified a fertilization (phot. 2, 3, 4) This methodology is based on high correlation between fruit setting and fertilization assessing in this way [10]. Data were analyzed using Stat program for Windows with t-Duncan test at α = 0.05.

Paraffin method. The aim of the investigation of microscopic preparations using the paraffin method was to determine the effect of NAA on the anatomical changes in plum fruits. For paraffin preparations of young fruits samples were taken every day beginning from the day before the NAA application up to the 8^th day after the treatment. Six young fruit (fruitlets) were prepared from combination every day. Specimens were fixed with Carnoy liquid and saturated with paraffin wax (54-56°C melting point). After slicing (5-7 µm of thickness) they were stained with Heidenhain haematoxylin [6] and observed in microscope (Hund Wetzlar H500).

RESULTS AND DISCUSSION

It was found that percentage of fertilized flowers (phot. 2) is exceeded in auxin treatments than in the control (tab. 1). These results suggested that stimulating effect of NAA on the fertilization is connected with ethylene production and characteristic of this gas [15, 16].

Microscopic studies using the fluorescence method showed an increased percentage of flowers with the chalazal part and vascular bundles of the ovule with callose after the auxin treatment (tab. 2). Vascular bundles are connected with the chalazal part of ovule (phot. 5). Bundles transport nutritive compounds to the nucellus and make possible its correct development (phot. 6-7). Callose deposited all chalazal part (phot. 8-9) and vascular bundles caused block assimilate flow and preventing the normal development of ovule.

The obtained data show that the blocking of the chalazal part of ovule with callose brings about the fruit drop (tab. 3). This finding agrees with data reported by Law and Yeung [9], who observed that the presence of callose in cell walls of the ovule is a symptom of its future degeneration. Mirza [13] observed a close connection between the development of seeds in dropped bean flowers and the deposition of callose in vascular bundles. The results of the presented experiment suggest that the chalazal part with callose blocks the inflow of assimilates bringing about the degeneration of the ovule and hence the premature fruit drop. Different authors also confirm these results. Dittmann and Stosser [5] found the presence of callose in the chalazal part of ovule of prematurely dropping fruits while Rodrigo and Herrero [18] and Pimienta and Polito [14] observed dying back ovules with callose in their chalazal part. These authors report that the deposition of callose within chalaza blocks the assimilate flow to the ovule, bringing about its degeneration. They also showed the decreasing content of carbohydrates in ovules after the chalazal part was blocked. However, it is still unclear if the synthesis of callose and deposition in chalazal part are the cause of the ovule degeneration or its result. According to Arbeloa and Herrero [2] the synthesis of callose precedes ovule degeneration. This agrees with the results of researches showing the presence of callose two days before distinct histological signs of the ovule degeneration [14]. The results of experiments by Aloni et al. [1] suggest that this auxin is not the direct factor inducing the synthesis of callose. They observed that exogenous NAA induced the removal of callose from the sieve tissue of shoots. If NAA does not directly influence the synthesis of callose, ethylene could be the responsible factor.

The inhibition of pro-embryo development also resulted from the inhibited growth of the ovule. The structure of embryo sac and endosperm was correctly developed in control (phot. 10). As first effects of exogenous auxin application, inhibition of endosperm development was observed (phot. 11). Next effects were observed on the second day after treatment. The inhibition of pro-embryo development was visible (phot. 12). Complete deformation of pro-embryo and the inhibition of endosperm development are visible on the third day after the NAA treatment (phot. 13). Additionally, granulation of endosperm was observed. Endosperm was lumpy and stuck together around wall of embryo sac.

Pro-embryo deformation could have resulted from endosperm inhibition [17]. Vitagliano et al. [22] reported similar results. They applied a solution of an NAA derivative and ethephone to peach trees. In microscopic preparations they found deformation of the ovule after the application of an NAA solution and the stunted growth and destruction of embryos after an ethephone treatment. These authors also found a rapid increase in NAA-induced ethylene. Byers et al. [3] reported a similarly destructive effect of synthetic auxin (2,4,5-TP). They recorded reduced numbers of seeds and a decrease in apple size. The results quoted above and these obtained in the presented experiment suggest that the destructive changes in the ovule are due to an increased ethylene concentration [7], however, it is difficult to state whether they are an intermediate or direct effect of its appearance. It is also difficult to state whether ethylene stimulates the production of callose, which blocks the chalazal part and brings about degeneration within the embryo sac, or directly affects the inhibition of endosperm and pro-embryo development, thus resulting in the synthesis of callose. In the presented experiment both types of symptoms were noted almost at the same time, that is 3-4 days after the NAA treatment.

AKNOWLEDGEMENTS

The research was supported by the State Committee for Scientific Research (KBN) grant No. G-1360/KS/01-02.

REFERENCES

1. Aloni R., Raviv A., Peterson C.A., 1991. The role of auxin in the removal of dormancy callose and resumption of phloem activity in Vitis vinifera. Can. J. Bot. 69,. 1825-1832.

2. Arbeloa A., Herrero M., 1985. Translocation to the ovule and female sterility in peach. Anales de la Estacion Experimental de Aula Dei. 17, 214-220.

3. Byers R.E., Yoder K.S., Mattus G.E., 1983. Reduction in russetting of ‘Golden Delicious’ apples with 2,4,5-TP and other compounds. Hort. Sci. 18, 63-65.

4. Curry E.A., 1991. NAA-induced ethylene and ACC in ‘Delicious’ spur tissues: changes with temperature and time. J. Amer. Soc. Hort. Sci. 116, 846-850.

5. Dittmann K., Stosser R., 1999. Development of ovules in relation to premature fruit drop in Prunus-species. Angewandte Botanik 73, 86-98.

6. Filutowicz A., Kużdowicz A., 1951. Mikrotechnika roslinna [Plant microtechnology]. PWN, Warszawa [in Polish].

7. Gonkiewicz A., Nosal K., 2006. Wydzielanie etylenu przez kwiaty i zawiazki owocowe sliw po zastosowaniu auksyny NAA (NAA-induced ethylene evolution by plum flowers and fruitlets). Folia Hort. 18/1, 63-72 [in Polish].

8. Kvale A., 1983. Chemical thinning of fruit in 1983. Gartneryrket. 73, 416.

9. Law S.K., Yeung E.C., 1989. Embryology of Calypso bulbosa. I. Ovule development. Am. J. Bot. 76, 1668-1674.

10. Lech W., 1980. Wybrane zagadnienia dotyczace kwitnienia i zawiazania owoców wisni [Select problems relating bloomings and fruit set of cherry]. Rozpr. hab. 77. Akademia Rolnicza w Krakowie [in Polish].

11. Lott R.V., Simons R.K., 1966. Sequential development of floral tube and style abscission in the Montmorency Cherry Prunus cerasus L. J. Amer. Soc. Hort. Sci. 88, 208-218.

12. Martin F.W., 1959. Staining and observing pollen tubes style by means of fluorescence. Stain Technol. 34, 125-128.

13. Mirza A.A., 1990. Photosynthate distribution, carbohydrate level in young pods, and viability of developing seeds in relation to abscission of reproductive structures in Phaseolus vulgaris L. Dissertation Abstracts International. B. Sci. Eng. 51, 513B.

14. Pimienta E., Polito V.S., 1982. Ovule abortion in ‘Nonpareil’ almond (Prunus dulcis [Mill.] D.A. Webb). Am. J. Bot. 69, 913-920.

15. Piskornik Z., 1986. The role of ethylene in the pollination and senescence of flower of bulbous plants. Acta Hort. 181, 407-413.

16. Piskornik Z., Mareczek A., Mazur A., 1989. Effect of ethylene and 1-aminocyclopropane-1-carboxylic acid on pollen germination of ornamental plants. Acta Physiol. Plant. 11, 365-371.

17. Rodkiewicz B., 1973. Embriologia roœlin kwiatowych [Embryology of flower plants]. PWN. Warszawa [in Polish].

18. Rodrigo J., Herrero M., 1998. Influence of intraovular reserves on ovule fate in apricot (Prunus armeniaca L.). Sexual Plant Repr. 11, 86-93.

19. Schönherr J., Baur P., Uhlig B.A., 2000. Rates of cuticular penetration of 1-naphthylacetic acid (NAA) as affected by adjuvants, temperature, humidity and water quality. Plant Growth Reg. 31, 61-74.

20. Wędzony M., 1996. Mikroskopia fruorescencyjna dla botaników [Fluorescence microscopy for botanists]. Monografie 5. Kraków [in Polish].

21. Williams K.M., Fallahi E., 1999. The effects of exogenous bioregulators and environment on regular cropping of apple. Proc. of ‘The riddle of regular cropping: the case for hormones, nutrition, exogenous bioregulators and environmental factors’. HortTechnology 9, 323-327.

22. Vitagliano C., Catania M., Andreucci A.C., Monet R., 1998. Possible effect of chemical thinners on seeds of Prunus persica cv. Redhaven. Acta Hort. 465, 655-661.

Accepted for print: 13.12.2006

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.